Amyloidosis is a serious disease caused by extracellular deposition of insoluble abnormal fibrils (Pepys, 2006). Systemic amyloidosis, with deposits in the viscera, blood vessels and connective tissue, is usually fatal, causing about one per thousand deaths in developed countries. About 25 different unrelated human proteins form amyloid fibrils in vivo. Amyloid is deposited when there is: (i) sustained exposure to either normal or increased concentrations of a normal, potentially amyloidogenic, protein; (ii) when an abnormal amyloidogenic protein is produced as a consequence of an acquired disease; or (iii) when a gene mutation encodes an amyloidogenic variant protein. Fibrillogenesis results from reduced stability of the native fold of the fibril precursor protein, so that under physiological conditions it populates partly unfolded intermediate states which aggregate as stable amyloid fibrils with the pathognomonic cross-β sheet core structure (Sunde et al 1997).
Wild type transthyretin, the normal plasma protein which transports thyroid hormone and retinol binding protein, is inherently amyloidogenic and forms microscopic amyloid deposits of uncertain clinical significance in all individuals aged over 80 years. Massive deposits in the heart can also occur, causing fatal senile cardiac transthyretin amyloidosis. The inherent amyloidogenicity of wild type transthyretin is markedly enhanced by most of the reported >80 different point mutations which encode single residue substitutions in the transthyretin sequence (Saraiva, 2002). These mutations cause autosomal dominant adult onset hereditary amyloidosis, a universally fatal condition affecting about 10,000 patients worldwide. The usual clinical presentation is familial amyloid polyneuropathy, with predominant peripheral and autonomic neuropathy, but there is commonly also serious involvement of the heart, kidneys and eyes. The condition typically presents after the causative gene has been transmitted to the proband's offspring, ensuring persistence of this devastating disease. Amyloidogenic mutations occur in all ethnic groups, but by far the most common, V30M, clusters in three geographical foci: Northern Portugal, Northern Sweden and parts of Japan. A common amyloidogenic variant in the UK and Eire is T60A. Transthyretin amyloidosis predominantly affecting the heart is particularly associated with the V122I variant, which is very rare in Caucasians but is carried by 4% of African Americans: 1.3 million people, including 13,000 individuals homozygous for the mutation (Jacobson, 1997). It is the second most common pathogenic mutation in that population after sickle cell haemoglobin. Cardiac transthyretin amyloidosis presents as progressive, ultimately fatal, heart failure due to restrictive cardiomyopathy, is rarely suspected and is usually misdiagnosed as coronary heart disease.
Liver transplantation provides an effective treatment for some patients with transthyretin amyloidosis. Transthyretin is synthesized by hepatocytes and by the choroid plexus. Liver transplantation removes the source of the amyloidogenic variant transthyretin in the plasma and replaces it with wild type transthyretin, however the procedure is available for only a minority of patients. There is a severe shortage of donor livers and the diagnosis of transthyretin amyloidosis is often too late for optimal results to be obtained. Patients with mutations other than V30M can develop rapidly progressive cardiac amyloidosis after transplantation. In patients with predominant cardiac amyloid, heart transplantation is a possible option, but most are too old and are not acceptable recipients for scarce donor organs. Furthermore liver transplantation does not affect production of variant amyloidogenic transthyretin by the choroid plexus and deposition of transthyretin amyloid in the eye and leptomeninges can thus progress despite disappearance of variant transthyretin from the circulation.
In view of the limitations of transplantation therapy, therapeutic drug approaches have been investigated. The native transthyretin molecule is very well characterised and is a homotetramer of molecular weight 55,044 Da, and the non-covalently associated protomers, of mass 13,761, each contain 127 residues with a β-sandwich fold. The native tetramer binds a single retinol binding protein molecule and contains two identical negatively cooperative L-thyroxine (T4) binding pockets. Amyloid fibril formation by transthyretin involves dissociation of the tetramer, partial unfolding of the protomers and then aggregation into the amyloid cross-β core structure.
One therapeutic drug approach is taught in WO03/013508 which describes agents comprising ligands capable of being bound by transthyretin which are covalently co-linked by a linker. The purpose of these agents is to form complexes between separate transthyretin tetramers in the subject to be treated. This approach relies on the complexes being recognised by the body as abnormal and rapidly cleared from the circulation. In this way, the amyloidogenic protein is no longer available as a source for amyloid deposition.
Another approach to the potential treatment of transthyretin amyloidosis has been to identify small molecule ligands which are specifically bound in the thyroid hormone binding pocket so as to stabilise the native transthyretin tetrameric structure and thereby prevent dissociation into dimers and protomers leading to fibrillogenic aggregation. This approach is described, for example, in WO2004/05635 where an array of biphenyl and benzoxazole compounds are described, including 2-(3,5-dichlorophenyl)benzo[d]oxazole-6-carboxylic acid (6). In the academic literature, in vitro studies have shown that bis-arylamine compounds have affinity for the binding pocket in transthyretin and can stabilise the tetrameric form. Oza et al (2002) describe the structures of various compounds of this type. In this paper, a distinction is made between a “forward mode” of binding and a “reverse mode”. Oza et al is particularly concerned with analogues of diclofenac (1), which has a ring bearing a chlorine substituent and a second ring bearing a carboxylate group. The diclofenac analogue, 2-(3,5-dichlorophenyl amino)benzoic acid (2) and diclofenac itself both bind in the “reverse mode” with the carboxylate bearing ring occupying the inner binding cavity of the transthyretin binding pockets. Wiseman et al (2005) also shows compound (2) binding in reverse mode. Green et al (2003) describe various bivalent inhibitors of transthyretin which comprise a pair of ligands linked together by a linker. When bound by the transthyretin tetramer, each ligand is situated in a binding pocket and the linker is situated in a central channel that runs through the transthyretin tetramer. The linker was therefore covalently attached to the aromatic group intended for binding to the inner binding cavity of the binding pockets. Whilst the authors found that these bivalent inhibitors stabilised tetrameric transthyretin reconstituted from dissociated protomers, these inhibitors were generally poor in the standard transthyretin fibril formation assay indicating that they were not bound at all by native tetrameric transthyretin. These results suggest that the approach of transthyretin stabilisation by bivalent compounds would not be successful in the treatment or prevention of transthyretin amyloidosis.